Abstract. Diabetes affects approximately 346 million people worldwide. In 2004, an estimated 3.4 million people died from the consequence of high blood glucose. The World Health Organization projects that diabetes death will double between 2005 and 2030. Both type 1 diabetes (T1D) and type 2 diabetes (T2D) result from the decrease of human pancreatic beta cell mass. After the diagnosis of high blood glucose, there are still residual beta cells detected in diabetes patients. The regeneration of human pancreatic beta cell mass will provide a novel approach for diabetes therapy. In healthy adult beta cell mass is maintained at a slow proliferation rate. However, adult pancreatic beta cell mass increases in certain conditions, such as obesity, pregnancy, etc. Thus, an easy and robust method to enhance pancreatic beta cell proliferation can be used as a new approach to treat diabetes patients. Cell-permeable compounds provide a convenient and efficient approach to control cell proliferation. Chemical screens have been applied to identify chemical inducers of pancreatic beta cell expansion. However, many compounds identified using rodents or animal models show little or moderator activities on primary human islets. In addition, most of the previous studies used healthy islet samples. It is not clear whether the identified compounds improve proliferation of T1D or T2D islets. In our previous studies, we have established several chemical screen platforms to identify the compounds promoting the generation, survival and function of human pluripotent stem cell (hPSC)-derived beta-like cells. Here, we propose to use hPSC-derived beta-like cells to screen for compounds that promote human pancreatic beta cell replication, and to validate the identified compounds using human healthy, T1D, T2D islets. In the preliminary studies, we have screened more than 1,200 compounds and identified hits that potentially increase the proliferation of hPSC-derived pancreatic beta-like cells. Some of hit compounds were further confirmed using primary human islets. Here, we proposed to expand the established screening platform to large scale and identify a list of ?chemical probe? using follow-up assays. Finally, we will study the mechanism of action of the chemical probes. The identified chemical probes can be directly used for anti-diabetes drug development or be used as the probes to discover novel targets for future drug discovery. To reach these goals, three aims were proposed, including 1) Carry out a high content screen to identify the compounds promoting human pancreatic beta cell proliferation; 2) Perform follow-up assays to finalize the ?chemical probe? list; 3) Study the mechanism of action of the compounds.